Regulator T cells: specific for antigen and/or antigen receptors?

Adaptive immune responses are regulated by many different molecular and cellular effectors. Regulator T cells are coming to their rights again, and these T cells seem to have ordinary alpha/beta T-cell receptors (TCRs) and to develop in the thymus. Autoimmune responses are tightly regulated by such regulatory T cells, a phenomenon which is beneficial to the host in autoimmune situations. However, the regulation of autoimmune responses to tumour cells is harmful to the host, as this regulation delays the defence against the outgrowth of neoplastic cells. In the present review, we discuss whether regulatory T cells are specific for antigen and/or for antigen receptors. Our interest in these phenomena comes from the findings that T cells produce many more TCR-alpha and TCR-beta chains than are necessary for surface membrane expression of TCR-alphabeta heterodimers with CD3 complexes. Excess TCR chains are degraded by the proteasomes, and TCR peptides thus become available to the assembly pathway of major histocompatibility complex class I molecules. Consequently, do T cells express two different identification markers on the cell membrane, the TCR-alphabeta clonotype for recognition by B-cell receptors and clonotypic TCR-alphabeta peptides for recognition by T cells?

Relative resistance to nasally induced tolerance in non-obese diabetic mice but not other I-A(g7)-expressing mouse strains.

I-A(g7) is a unique class II MHC molecule that is clearly associated with autoimmune diabetes in non-obese diabetic (NOD) mice. To determine if I-A(g7) is defective in its ability to deliver tolerogenic signals in vivo, H-2(g7) mice were nasally pretreated with antigen, prior to immunization, to induce antigen-specific regulation. Nasally pretreated NOR (H-2(g7)) and (NON).NOD (H-2(g7)) congenic mice showed responses similar to those of NON (H-2(nb1)), BALB/c (H-2(d)) and B10.PL (H-2(u)) mice-a reduced recall response and a deviated T(h) cytokine profile. However, we found that NOD (H-2(g7)) mice are comparatively resistant to immunological tolerance induced by nasal pretreatment, such that at the usually effective dose no significant reduction was seen in the proliferative recall responses to nominal antigen after immunization. (NOD x BALB/c)F(1) (H-2(g7/d)) and (NOD x NOR)F(1) (H-2(g7)) mice were similarly resistant to nasal-induced tolerance, although significantly higher nasal doses of antigen were able to overcome the resistance in NOD and F(1) mice. Interestingly, activated NOD T cells were resistant to cell death induced by re-stimulation with plate-bound anti-CD3. These results demonstrate that activated T cells in NOD mice are defective in their ability to respond to regulatory signals delivered in vivo or in vitro. Furthermore, NOD T cells have an increased resistance to tolerance induced by I-A(g7)-dependent (antigen) or I-A(g7)-independent (anti-CD3) mechanisms. Thus, while I-A(g7) may contribute to insulin-dependent diabetes mellitus by selecting a particular repertoire of self-reactive T cell clones, additional defects in the peripheral T cells themselves are required to allow the expansion of diabetogenic clones and the development of autoimmune disease.

MHC class I-restricted determinants on the glutamic acid decarboxylase 65 molecule induce spontaneous CTL activity.

CD4(+) T cell responses to glutamic acid decarboxylase (GAD65) spontaneously arise in nonobese diabetic (NOD) mice before the onset of insulin-dependent diabetes mellitus (IDDM) and may be critical to the pathogenic process. However, since both CD4(+) and CD8(+) T cells are involved in autoimmune diabetes, we sought to determine whether GAD65-specific CD8(+) T cells were also present in prediabetic NOD mice and contribute to IDDM. To refine the analysis, putative K(d)-binding determinants that were proximal to previously described dominant Th determinants (206-220 and 524-543) were examined for their ability to elicit cytolytic activity in young NOD mice. Naive NOD spleen cells stimulated with GAD65 peptides 206-214 (p206) and 546-554 (p546) produced IFN-gamma and showed Ag-specific CTL responses against targets pulsed with homologous peptide. Conversely, several GAD peptides distal to the Th determinants, and control K(d)-binding peptides did not induce similar responses. Spontaneous CTL responses to p206 and p546 were mediated by CD8(+) T cells that are capable of lysing GAD65-expressing target cells, and p546-specific T cells transferred insulitis to NOD.scid mice. Young NOD mice pretreated with p206 and p546 showed reduced CTL responses to homologous peptides and a delay in the onset of IDDM. Thus, MHC class I-restricted responses to GAD65 may provide an inflammatory focus for the generation of islet-specific pathogenesis and beta cell destruction. This report reveals a potential therapeutic role for MHC class I-restricted peptides in treating autoimmune disease and revisits the notion that the CD4- and CD8-inducing determinants on some molecules may benefit from a proximal relationship.

Environmental modulation of autoimmune arthritis involves the spontaneous microbial induction of T cell responses to regulatory determinants within heat shock protein 65.

Both genetic and environmental factors are believed to be involved in the induction of autoimmune diseases. Adjuvant arthritis (AA) is inducible in susceptible rat strains by injection of Mycobacterium tuberculosis, and arthritic rats raise T cell responses to the 65-kDa mycobacterial heat-shock protein (Bhsp65). We observed that Fischer 344 (F344) rats raised in a barrier facility (BF-F344) are susceptible to AA, whereas F344 rats maintained in a conventional facility (CV-F344) show significantly reduced incidence and severity of AA, despite responding well to the arthritogenic determinant within Bhsp65. The acquisition of protection from AA can be circumvented if rats are maintained on neomycin/acidified water. Strikingly, naive unimmunized CV-F344 rats but not BF-F344 rats raised T cell responses to Bhsp65 C-terminal determinants (BCTD) (we have previously shown that BCTD are involved in regulation of acute AA in the Lewis rat); however, T cells of naive CV-F344 and BF-F344 gave a comparable level of proliferative response to a mitogen, but no response at all to an irrelevant Ag. Furthermore, adoptive transfer into naive BF-F344 rats of splenic cells of naive CV-F344 rats (restimulated with BCTD in vitro) before induction of AA resulted in a considerably reduced severity of AA. These results suggest that spontaneous (inadvertent) priming of BCTD-reactive T cells, owing to determinant mimicry between Bhsp65 and its homologues in microbial agents in the conventional environment, is involved in modulating the severity of AA in CV-F344 rats. These results have important implications in broadening understanding of the host-microbe interaction in human autoimmune diseases.

Regulatory and effector CD4 T cells in nonobese diabetic mice recognize overlapping determinants on glutamic acid decarboxylase and use distinct V beta genes.

The 524--543 region of glutamic acid decarboxylase (GAD65), GAD65(524--543), is one of the first fragments of this islet Ag to induce proliferative T cell responses in the nonobese diabetic (NOD) mouse model of spontaneous autoimmune diabetes. Furthermore, NOD mice given tolerogenic doses of GAD65(524--543) are protected from spontaneous and cyclophosphamide-induced diabetes. In this study, we report that there are at least two I-A(g7)-restricted determinants present in the GAD65(524--543) sequence, each capable of recruiting unique T cell repertoires characterized by distinct TCR V beta gene use. CD4(+) T cells arise spontaneously in young NOD mice to an apparently dominant determinant found within the GAD65 peptide 530--543 (p530); however, T cells to the overlapping determinant 524-538 (p524) dominate the response only after immunization with GAD65(524--543). All p530-responsive T cells used the V beta 4 gene, whereas the V beta 12 gene is preferentially used to encode the TCR of p524-responsive T cell populations. T cell clones and hybridomas from both of these T cell groups were responsive to APC pulsed with GAD65(524--543) or whole rGAD65. p524-reactive cells appeared to be regulatory upon adoptive transfer into young NOD mice and could inhibit insulin-dependent diabetes mellitus development, although they were unable to produce IL-4, IL-10, or TGF beta upon antigenic challenge. Furthermore, we found that i.p. injection with p524/IFA was very effective in providing protection from cyclophosphamide-induced insulin-dependent diabetes mellitus. These data demonstrate that the regulatory T cells elicited by immunizing with GAD65(524--543) are unique and distinct from those that arise from spontaneous endogenous priming, and that T cells to this limited region of GAD65 may be either regulatory or pathogenic.

Molecular characterization of the T cell repertoire using immunoscope analysis and its possible implementation in clinical practice.

T lymphocytes play a central role in the pathogenesis of a large number of human conditions including autoimmunity and graft rejection. Although T cells are key players in mounting immune responses, the assessment of T cell repertoires has yet to find an important role in clinical decision making. In this review, we discuss the "immunoscope" technique and its potential diagnostic role in a variety of clinical scenarios. This is an RT-PCR based approach that subdivides a bulk T cell population (i. e. from blood, lymph, spleen, or tissue) into approximately 2800 groups based upon rearranged variable beta (Vbeta)/joining beta (Jbeta) gene segments and the resulting length of the T cell receptor's (TCR's) third complementarity determining region (CDR-3). This extensive subdivision, or focusing, allows clonal expansions to be directly observed. Such a fine-tuned analysis has revealed previously unappreciated aspects of the T cell repertoire. For instance, an antigen-specific immune response can be divided into both public and non-public components. The non-public repertoire contains the majority of the expanding T cells which are unique to the individual (private), or shared by only some (semi-private), while "public" T cells can be found responding to the antigenic determinant in every individual. Although they are often a minority of the response, the public T cell repertoire seems to play a more important role in defining, as well as driving, the overall immune phenotype in the animal. Immunoscope analysis has identified public and non-public responses in human pathologies, such as multiple sclerosis. The ability to characterize the driver T cells dictating the state of immunity/autoimmunity in individual patients will be an important step towards understanding autoimmunity and designing effective treatment for a variety of conditions including rheumatoid arthritis and multiple sclerosis. We review the current literature involving public and non-public repertoires and discuss the prospect that immunoscope analysis may play a central role in the study and perhaps the management of human autoimmune diseases, and cancer.

Immunogenicity of self antigens is unrelated to MHC-binding affinity: T-cell determinant structure of Golli-MBP in the BALB/c mouse.

The 'classical' myelin basic protein (MBP) exons belong to a much larger unit, termed the 'Golli-MBP' gene. Here we have examined the T-cell determinant structure of the Golli protein region in the BALB/c mouse. Golli p10-24, which was shown to have the strongest affinity for I-A(d), could not induce T-cell activation. Paradoxically, the poorer binding, overlapping p5-19 was effective at inducing T-cell proliferation. Thus, immunogenicity is not necessarily related to the MHC-binding affinity of self-peptides. In addition, MBP: p151-168-specific T cell clones responded only poorly to J37, a Golli-MBP protein, while MBP: 59-76-specific clones responded well to J37.

Residual public repertoires to self.

The consensus view about the constitution of the T cell receptor repertoire has shifted greatly even during this decade. Although the discovery of autoimmunity in the fifties had clearly shown that a repertoire must exist directed against self antigens, the extent of this repertoire was not fully appreciated. In our work we have tried to elucidate the nature of the antigenic specificities against which this self-directed repertoire is directed. The non-tolerized (residual) self-directed repertoire is a direct consequence of the hierarchy of antigenic determinant display, and is the most important influence in the organism's choice of which T cells to delete. Certain determinants remain "silent" and are neither displayed in the thymus nor in the periphery: these are a heterogeneous group which are invisible to T cells for a variety of reasons. One reason relates to the processing and presentation of determinants, and a second derives from the nature of the T cell receptor (TcR) and the avidity of the T cell for its target specificity.

Cutting edge: introduction of an endopeptidase cleavage motif into a determinant flanking region of hen egg lysozyme results in enhanced T cell determinant display.

The choice of which determinants of a whole Ag will be presented on cell surface MHC class II molecules after uptake and processing by APC is the result of the interplay between structural characteristics of the Ag and the processing machinery of the APC. In this study, we demonstrate that introduction of a dibasic motif adjacent to a subdominant determinant enhances the presentation of this determinant from the whole molecule. This is the first report showing that a single amino acid substitution in a whole Ag, designed to introduce an endopeptidase recognition site, enhances display of class II-restricted determinants, most likely by creating a peptide chain cleavage in the antigenic molecule. Our findings have important implications for the understanding of immunodominance and for vaccine design.

T cell receptor complementarity determining region 3 length analysis reveals the absence of a characteristic public T cell repertoire in neonatal tolerance. The response in the "tolerant" mouse within the residual repertoire is quantitatively similar but qualitatively different.

All adult BALB/c mice immunized with hen egg white lysozyme (HEL) or its dominant determinant, peptide (p)106-116, mount a T cell response using a "public" Vbeta8.2Jbeta1.5 T cell clone. Neonatal exposure to tolerance-inducing doses of antigen can drastically diminish responsiveness in the draining lymph nodes but not in the spleens of animals challenged as adults with the cognate antigen. To determine the role of T cell deletion or anergy within the mechanisms of observed neonatal "tolerance," we treated neonatal BALB/c mice with HEL and directly followed the characteristic public clone using complementarity determining region 3 length T cell repertoire analysis. Our results confirm that despite intraperitoneal injection of neonates with a high dose of HEL emulsified in incomplete Freund's adjuvant, a strong splenic proliferative response to HEL was observed upon recall. However, the adult splenic T cell response of these neonatally treated mice lacked the usual Vbeta8.2Jbeta1.5 public clone characteristic of HEL-primed BALB/c mice. After challenge with HEL-complete Freund's adjuvant as adults, immunoglobulin (Ig)G2a isotype antibody was drastically reduced, and IgG1 was found to be the predominant anti-HEL IgG isotype expressed, indicating a deviation of cytokine response toward T helper type 2. 5-wk-old mice, nasally instilled with tolerogenic doses of HEL p106-116, also showed significant inhibition of this public T cell expansion. These results demonstrate that during neonatal and adult nasal tolerance induction, deletion/anergy removes the public clone, exposing a response of similar specificity but that is characterized by the T helper type 2 phenotype and a splenic residence.

Processing and reactivity of T cell epitopes containing two cysteine residues from hen egg-white lysozyme (HEL74-90).

The Ag processing and structural requirements involved in the generation of a major T cell epitope from the hen egg-white lysozyme protein (HEL74-88), containing two cysteine residues at positions 76 and 80, were investigated. Several T cell hybridomas derived from both low responder (I-Ab) and high responder (I-Ak) mice recognize this region. These hybridomas are strongly responsive to native HEL, but unresponsive to the reduced and carboxymethylated protein. Air-oxidized HEL74-88 peptide was unable to bind I-Ak molecules and failed to stimulate T cells in the absence of intracellular Ag processing. Further functional competition assays showed that alkylation of cysteine residues with bulky methyl groups interferes with the contacts for the MHC class II molecules (I-Ak) of high responder mice and the I-Ab-restricted TCR of low responder mice. Serine substitutions of the cysteine residues of HEL74-88 either enhanced or abrogated T cell stimulation by the peptides without significant alterations in the class II binding. These results suggest that the cysteine residues of peptides must be free from disulfide bonding for efficient stimulation of T cells and yet frequently used modifications of cysteine residues may not be suitable for peptide-based vaccine development.

The self-directed T cell repertoire: its creation and activation.

The considerable breadth of the self-directed T cell repertoire has only fully been appreciated during this past decade. It is a potential repertoire which can be tapped in various ways, most evidently in the study of autoimmune diseases, when because of a variety of factors, there is enhanced processing and presentation of determinants on self antigens. In this review, we have focused on the engagement of this self-reactive repertoire and some of the rules involved, which are not always so obvious. The total "residual" self-reactive repertoire directed against a single antigen (that remains after negative selection) will be a heterogeneous assemblage of T cells - (a) high affinity T cells directed against determinants whose presentation during tolerance induction was prevented, eg. through competitive binding by neighboring determinants; (b) lower affinity T cells directed against well-presented (dominant), as well as poorly-presented (cryptic) determinants; and (c) high affinity T cells directed against poorly-presented determinants, which are only presented during inflammation. Under conditions that favor upregulation of previously cryptic self determinants, one or more of the above subsets of the 'protected' T cell repertoires can be stimulated by these self determinants, leading to induction of autoreactivity. The latter could eventually result in autoimmunity under permissive conditions governed by MHC and non-MHC genes. Interestingly, the very same repertoires that appear to be recruited into pathogenic autoimmune destruction may be alternatively manipulated as a source of anti-cancer treatment. It is now evident that many tumor antigens are unmutated self antigens, and cryptic determinants within such tumor antigens could be used to recruit the anticryptic T cell repertoire for induction of anti-tumor immunity.

The self-directed T cell repertoire against mouse lysozyme reflects the influence of the hierarchy of its own determinants and can be engaged by a foreign lysozyme.

The T cell repertoire is shaped by the processes of positive and negative selection. We have previously shown that mice are tolerant to a native self-Ag, mouse lysozyme (ML), but they respond vigorously when challenged with different ML peptides ("cryptic" self-determinants). In this study, we have addressed the issue of the physiological significance of both the hierarchy (dominance/crypticity) of self-determinants within ML and the anti-cryptic, self (ML)-directed T cell repertoire. Our results demonstrate that there are several ML peptides that bind well to MHC but are totally nonimmunogenic when tested for proliferative T cell response and cytokine secretion: a subset of these peptides presumably represent the originally dominant self-determinants of ML, which have rendered the T cells tolerant during thymic selection. Other ML peptides, which bind well to MHC and are immunogenic, correspond to the cryptic determinants of ML: T cells against cryptic ML determinants escape tolerance induction. Thus, the mature T cell repertoire against ML bears the direct imprint of the hierarchy of self (ML)-determinants. Interestingly, hen egg white lysozyme could prime T cells in vivo that were cross-reactive with certain cryptic ML determinants, and vice versa, without requiring any coimmunization with the foreign lysozyme and ML peptide(s). Moreover, repeated, deliberate priming and expansion of T cells by hen egg white lysozyme immunization concomitantly enhanced T cell response to such cross-reactive ML determinants. This reciprocal self-foreign determinant cross-reactivity may play a previously unrecognized, but crucial, role in the expansion and diversification of self-reactive clones in the autoimmune response.

Oligodendrocyte-specific protein peptides induce experimental autoimmune encephalomyelitis in SJL/J mice.

Oligodendrocyte-specific protein (OSP) is a recently isolated and cloned, 207-aa, hydrophobic, four-transmembrane protein found in CNS myelin. It represents approximately 7% of total myelin protein. The OSP cDNA sequence has no significant homology with previously reported genes, but the predicted protein structure suggests that OSP is a CNS homologue of peripheral myelin protein-22. We previously reported the presence of anti-OSP Abs in the cerebrospinal fluid of relapsing-remitting multiple sclerosis (MS) patients, but not control patient groups. In this study, we tested the ability of a panel of 20-mer peptides with 10-aa overlaps, representing the sequence of murine OSP, to induce experimental autoimmune encephalomyelitis (EAE), an animal model for MS. SJL mice challenged with murine OSP peptides 52-71, 82-101, 102-121, 142-161, 182-201, and 192-207 exhibited clinical EAE. OSP:52-71 elicited severe relapsing-remitting EAE in some individuals. All other encephalitogenic peptides elicited, at most, a loss of tail tonicity from which the mice most often completely recovered. Mononuclear cell infiltrates and focal demyelination characteristic of EAE were evident. T cell proliferative responses were seen with all encephalitogenic peptides except 142-161 and 182-201. OSP peptides 72-91 and 132-151 did not cause clinical EAE, but did elicit robust proliferative responses. B10.PL and PL/J mice challenged with the same OSP peptide doses as SJL mice did not exhibit clinical EAE. These results in the SJL EAE model, together with the results from MS patient clinical samples, make OSP a promising candidate for autoantigenic involvement in MS.

A single intramuscular injection with an adenovirus-expressing IL-12 protects BALB/c mice against Leishmania major infection, while treatment with an IL-4-expressing vector increases disease susceptibility in B10.D2 mice.

Experimental infection of the susceptible BALB/c (H-2d) mouse with the intracellular parasite Leishmania major induces a predominant Th2-type T cell response that eventually leads to death. In contrast, the resistant B10.D2 (H-2d) strain develops Th1 cells that control parasite replication and disease. In this study, we tested the ability of a recombinant adenovirus vector-expressing IL-12 to skew the immune response in a Th1 direction and prevent leishmaniasis in susceptible mice. We report that BALB/c mice treated with the Ad5IL-12 vector on the same day as parasitic challenge are significantly protected against leishmaniasis and acquired long-lasting immunity, because upon rechallenge with L. major parasites they were resistant to disease. The vector-derived IL-12 expression was transient and highly localized to the tissue after i.m. injection; it caused an increase in the number of Ag-specific IFN-gamma-secreting lymphocytes and enhanced NK cell activity in the draining popliteal node. In contrast, resistant B10.D2 mice given i.m. injections with a recombinant adenovirus-expressing IL-4 displayed greater susceptibility to disease, and severe lesions were produced in some of the infected animals. These results suggest the potential use of recombinant adenoviruses expressing cytokines as potent immunomodulatory agents for the generation of protective immune responses against intracellular pathogens.

Heterogeneity of the T cell response to immunodominant determinants within hen eggwhite lysozyme of individual syngeneic hybrid F1 mice: implications for autoimmunity and infection.

Hybrid F1 mice derived from inbred parental mouse strains are extensively used as animal models of human autoimmune diseases and transplantation. It is generally believed that with regard to immunologic studies, hybrid F1 mice behave in a consistent manner, equivalent to any other inbred mouse strain. In this study, we report that in comparison to inbred parental strains, individual hybrid F1 mice revealed a broad heterogeneity of proliferative response to the immunodominant determinants within hen eggwhite lysozyme (HEL). Of five parental strains tested, individual mice of three strains responding to only a few dominant HEL determinants (B6, BALB/c, and B10.PL) showed quite homogeneous patterns of response, whereas two mouse strains responsive to several determinants of HEL revealed either relative homogeneity (CBA/J mice) or heterogeneity (SJL mice) of response. However, in SJL mice, responses to major, dominant determinants of HEL were quite consistent. On the contrary, regardless of the consistency of response of parental strains, all three of F1 mice [[B6 x BALB/c]F1, [B6 x CBA/J]F1, and [SJL x B10.PL]F1] revealed significantly greater heterogeneity of response, which even involved the major, dominant determinants of HEL. We attribute the above heterogeneity of response to the competitive as well as aleatory nature of the interaction between various factors, including the coexistence of different MHC (parental as well as hybrid MHC) molecules, determinant capture, and the T cell repertoire. These results have important implications for studies on autoimmunity, infection, and vaccine design in human populations, where heterozygosity is the norm rather than the exception.

Recognition of prostate-specific antigenic peptide determinants by human CD4 and CD8 T cells.

It is now becoming accepted that one is not tolerant to all the determinants of self proteins: the T cell repertoire directed to some sequences in self proteins is intact and can be activated. When a self protein is exclusively expressed by tumour cells, the T cell repertoire directed to the particular self antigen can potentially be activated to attack the tumour: this would amount to induction of a beneficial autoimmune response. Prostate cancer offers a unique opportunity for activation of a tumour-specific immune response owing to the exclusive synthesis of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) by prostatic tissue and prostate tumour cells. In this study we examine the CD4 and CD8 T cell repertoires specific for peptides of PSA and PSM in normal human male individuals, using short-term, peptide antigen-driven CD4 and CD8 T cell lines. We show that short-term, CD4 T cell lines derived from six HLA-DR4 individuals showed strong proliferative responses to six of 10 tested peptides of PSA, selected as to contain a DR4 binding motif. Short-term, CD8 T cell lines from three HLA-A1 individuals showed specific cytolytic activity for autologous targets loaded with five of five tested peptides of PSA and PSM, selected to possess an HLA-A1 binding motif. One of the peptides chosen is termed a 'dual-motif' peptide, as it encodes determinants for both CD4 and CD8 T cells. These results, indicating the existence of CD4 and CD8 T cells against determinants of the self proteins, PSA and PSM, in healthy male individuals reveal the potential of the T cell repertoire from the typical prostate cancer patient to eradicate prostate tumours upon being appropriately activated.